Nucleic acid complex pair, competitive construct, and pcr kit using the same

Abstract

According to an exemplary embodiment of the present application, provided is a nucleic acid complex pair, which includes a first nucleic acid complex including a first determinant and a second tag; and a second nucleic acid complex including a second determinant and a second tag, wherein the first determinant includes a forward primer for first target DNA, the second determinant includes a reverse primer for the first target DNA, the first tag includes a base sequence complementary to the base sequence of the second tag, and the second tag includes a base sequence complementary to the base sequence of the first tag.

1 . A nucleic acid complex pair, the nucleic acid complex pair comprising:
 (a) a first nucleic acid complex,
 wherein the first nucleic acid complex comprises a first nucleic acid, a second nucleic acid, a first label, and a first linker, 
 wherein a 5′ end of the first nucleic acid is linked to a 3′ end of the second nucleic acid by the first linker, 
 wherein a 5′ end of the second nucleic acid is conjugated to the first label by a covalent bond, 
 wherein the first nucleic acid comprises a forward primer of a first target, 
 wherein the second nucleic acid comprises a first portion, 
 wherein the first portion has a first sequence; and 
   (b) a second nucleic acid complex,
 wherein the second nucleic acid complex comprises a third nucleic acid, a fourth nucleic acid, a second label, and a second linker, 
 wherein a 5′ end of the third nucleic acid is linked to a 3′ end of the fourth nucleic acid by the second linker, 
 wherein a 5′ end of the fourth nucleic acid is conjugated to the second label by a covalent bond, 
 wherein the third nucleic acid comprises a reverse primer of the first target, 
 wherein the fourth nucleic acid comprises a second portion, 
 wherein the second portion has a second sequence, 
 wherein the second sequence is complementary to the first sequence, 
   wherein the first label is one of a first signal substance-second signal substance pair, and the second label is the other of the first signal substance-second signal substance pair,   wherein a fluorescence intensity emitted from the first signal substance is adjusted by the second signal substance when the second nucleic acid binds to the fourth nucleic acid.   
     
     
         2 . The nucleic acid complex pair of  claim 1 ,
 wherein the first signal substance is a fluorophore,   wherein the second signal substance is a quencher,   wherein when the second signal substance is located within an effective distance from the first signal substance, the fluorophore interacts with the quencher, and   wherein the fluorescence intensity emitted from the first signal substance is adjusted by the second signal substance via an interaction between the fluorophore and the quencher.   
     
     
         3 . The nucleic acid complex pair of  claim 1 ,
 wherein the first signal substance is a first fluorophore,   wherein the second signal substance is a second fluorophore,   wherein when the second signal substance is located within an effective distance from the first signal substance, the second signal substance interacts with the first signal substance,   wherein an interaction between the first signal substance and the second signal substance is a förster resonance energy transfer(FRET), and   wherein the first signal substance is a donor, and the second signal substance is an acceptor.   
     
     
         4 . The nucleic acid complex pair of  claim 1 ,
 wherein the first portion is composed of an oligonucleotide consisting of 6 to 20 nucleotides, and   wherein the second portion is composed of an oligonucleotide consisting of 6 to 20 nucleotides.   
     
     
         5 . The nucleic acid complex pair of  claim 1 ,
 wherein the second nucleic acid comprises the first portion and a third portion, and the third portion has a third sequence,   wherein the fourth nucleic acid comprises the second portion and a fourth portion, and the fourth portion has a fourth sequence, and   wherein the third sequence is not complementary to the fourth sequence.   
     
     
         6 . The nucleic acid complex pair of  claim 5 ,
 wherein the first portion is located closer to the 5′ end of the second nucleic acid than the third portion.   
     
     
         7 . The nucleic acid complex pair of  claim 5 ,
 wherein the third portion is located closer to the 5′ end of the second nucleic acid than the first portion.   
     
     
         8 . The nucleic acid complex pair of  claim 1 ,
 wherein the first linker is a PCR blocker, and   wherein the second linker is a PCR blocker.   
     
     
         9 . The nucleic acid complex pair of  claim 2 ,
 wherein the forward primer of the first target is corresponding to a fifth portion of the first target,   wherein the reverse primer of the first target is corresponding to a sixth portion of the first target, and   wherein a distance between the fifth portion of the first target and the sixth portion of the first target is longer than the effective distance.   
     
     
         10 . A kit comprising:
 (a) a first nucleic acid complex pair,
 wherein the first nucleic acid complex pair comprising a first nucleic acid complex and a second nucleic acid complex, 
 wherein the first nucleic acid complex comprises a first nucleic acid, a second nucleic acid, a first label, and a first linker, 
 wherein a 5′ end of the first nucleic acid is linked to a 3′ end of the second nucleic acid by the first linker, 
 wherein a 5′ end of the second nucleic acid is conjugated to the first label by a covalent bond, 
 wherein the first nucleic acid comprises a forward primer of a first target, 
 wherein the second nucleic acid comprises a first portion, 
 wherein the first portion has a first sequence, 
 wherein the second nucleic acid complex comprises a third nucleic acid, a fourth nucleic acid, a second label, and a second linker, 
 wherein a 5′ end of the third nucleic acid is linked to a 3′ end of the fourth nucleic acid by the second linker, 
 wherein a 5′ end of the fourth nucleic acid is conjugated to the second label by a covalent bond, 
 wherein the third nucleic acid comprises a reverse primer of the first target, 
 wherein the fourth nucleic acid comprises a second portion, 
 wherein the second portion has a second sequence, and the second sequence is complementary to the first sequence, 
   wherein the first label is one of a first signal substance-second signal substance pair, and the second label is the other of the first signal substance-second signal substance pair,   wherein a fluorescence intensity emitted from the first signal substance is adjusted by the second signal substance when the second nucleic acid binds to the fourth nucleic acid; and   (b) a second nucleic acid complex pair,
 wherein the second nucleic acid complex pair comprising a third nucleic acid complex and a fourth nucleic acid complex, 
 wherein the third nucleic acid complex comprises a fifth nucleic acid, a sixth nucleic acid, a third label, and a third linker, 
 wherein a 5′ end of the fifth nucleic acid is linked to a 3′ end of the sixth nucleic acid by the third linker, 
 wherein a 5′ end of the sixth nucleic acid is conjugated to the third label by a covalent bond, 
 wherein the fifth nucleic acid comprises a forward primer of a second target, 
 wherein the sixth nucleic acid comprises a third portion, 
 wherein the third portion has a third sequence, 
 wherein the fourth nucleic acid complex comprises a seventh nucleic acid, an eighth nucleic acid, a fourth label, and a fourth linker, 
 wherein a 5′ end of the seventh nucleic acid is linked to a 3′ end of the eighth nucleic acid by the fourth linker, 
 wherein a 5′ end of the eighth nucleic acid is conjugated to the fourth label by a covalent bond, 
 wherein the seventh nucleic acid comprises a reverse primer of the second target, 
 wherein the eighth nucleic acid comprises a fourth portion, 
 wherein the fourth portion has a fourth sequence, and the fourth sequence is complementary to the third sequence, 
   wherein the third label is one of a third signal substance-fourth signal substance pair, and the fourth label is the other of the third signal substance-fourth signal substance pair, and   wherein a fluorescence intensity emitted from the third signal substance is adjusted by the fourth signal substance when the sixth nucleic acid binds to the eighth nucleic acid.   
     
     
         11 . The kit of  claim 10 ,
 wherein the first signal substance is a first fluorophore,   wherein the second signal substance is a first quencher,   wherein when the second signal substance is located within a first effective distance from the first signal substance, the first fluorophore interacts with the first quencher,   wherein the fluorescence intensity emitted from the first signal substance is adjusted by the second signal substance via an interaction between the first fluorophore and the first quencher,   wherein the third signal substance is a second fluorophore,   wherein the fourth signal substance is a second quencher, and   wherein when the third signal substance is located within a second effective distance from the fourth signal substance, the second fluorophore interacts with the second quencher, and   wherein the fluorescence intensity emitted from the third signal substance is adjusted by the fourth signal substance via an interaction between the second fluorophore and the second quencher.   
     
     
         12 . The kit of  claim 10 ,
 wherein the first sequence of the first portion is different from the third sequence of the third portion,   wherein the first sequence of the first portion is different from the fourth sequence of the fourth portion,   wherein a wavelength band of light emitted from the first signal substance is substantially the same as a wavelength band of light emitted from the third signal substance.   
     
     
         13 . The kit of  claim 10 ,
 wherein the first sequence of the first portion is different from the third sequence of the third portion,   wherein the first sequence of the first portion is different from the fourth sequence of the fourth portion,   wherein the first signal substance is composed of a same material as the third signal substance.   
     
     
         14 . The kit of  claim 10 ,
 wherein a wavelength band of light emitted from the first signal substance is substantially different from a wavelength band of light emitted from the third signal substance.   
     
     
         15 . The kit of  claim 10 ,
 wherein the first portion is composed of an oligonucleotide consisting of 6 to 20 nucleotides,   wherein the second portion is composed of an oligonucleotide consisting of 6 to 20 nucleotides,   wherein the third portion is composed of an oligonucleotide consisting of 6 to 20 nucleotides, and   wherein the fourth portion is composed of an oligonucleotide consisting of 6 to 20 nucleotides.   
     
     
         16 . The kit of  claim 10 ,
 wherein a complementary binding between the first portion and the second portion is dissociated at a first disassociation temperature, and a complementary binding between the third portion and fourth portion is dissociated at a second disassociation temperature, and   wherein the first dissociation temperature is higher than the second dissociation temperature.   
     
     
         17 . The kit of  claim 10 ,
 wherein the first target is associated with a first disease, and   wherein the second target is associated with a second disease different from the first disease.   
     
     
         18 . The kit of  claim 10 ,
 wherein the first linker is a PCR blocker,   wherein the second linker is a PCR blocker,   wherein the third linker is a PCR blocker, and   wherein the fourth linker is a PCR blocker.   
     
     
         19 . The kit of  claim 10 ,
 wherein the second nucleic acid comprises the first portion and a fifth portion, and the fifth portion has a fifth sequence,   wherein the fourth nucleic acid comprises the second portion and a sixth portion, and the sixth portion has a sixth sequence, and   wherein the fifth sequence is not complementary to the sixth sequence,   wherein the sixth nucleic acid comprises the third portion and a seventh portion, and the seventh portion has a seventh sequence,   wherein the eighth nucleic acid comprises the fourth portion and an eighth portion, and the eighth portion has an eighth sequence, and   wherein the seventh sequence is not complementary to the eighth sequence.   
     
     
         20 . The kit of  claim 11 ,
 wherein the forward primer of the first target is corresponding to a ninth portion of the first target,   wherein the reverse primer of the first target is corresponding to a tenth portion of the first target, and   wherein a distance between the ninth portion of the first target and the tenth portion of the first target is longer than the first effective distance,   wherein the forward primer of the second target is corresponding to an eleventh portion of the second target,   wherein the reverse primer of the second target is corresponding to a twelfth portion of the second target, and   wherein a distance between the eleventh portion of the second target and the twelfth portion of the second target is longer than the second effective distance.